Long-term blast control in high eating quality rice using multilines

Combining genetic heterogeneity and crop homogeneity serves a dual purpose: disease control and maintaining harvest quality. Multilines, which consist of a genetically uniform mixture of plants, have the potential to suppress disease while maintaining eating quality, yet practical methods that facilitate commercial use over large geographical areas are lacking. Here, we describe effective rice multiline management based on seed mixture composition changes informed by monitoring virulent blast races in Niigata Prefecture, Japan. The most elite nonglutinous cultivar, Koshihikari, was converted into the multiline, Koshihikari BL (blast resistant lines) and planted on 94,000 ha in 2005. The most destructive rice disease, blast, was 79.4% and 81.8% less severe in leaves and panicles, respectively, during the 2005–2019 period compared to the year 2004. In addition, fungicidal application was reduced by two-thirds after the introduction of BL. Our results suggest that seed mixture diversification and rotation of resistant BL provides long-term disease control by avoiding virulent race evolution.

www.nature.com/scientificreports/ Pia and Pii genes, were always mixed at a ratio of 1:2, but the composition of resistant ILs, containing Pita-2, Piz, Pib, Piz-t, and Pit genes, was changed every two to three years to avoid the breakdown of resistance 6 . These changes were determined by annually monitoring blast race distributions. In Niigata Prefecture, the predominant 5 blast races distributed from 1994 to 2004 were 001.0 (virulent to Koshihikari [Pik-s]), 003.0 (virulent to Pik-s and Pia), 005.0 (virulent to Pik-s and Pii), 007.0 (virulent to Pik-s, Pia, and Pii), and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) (Fig. 2a, Supplementary Table S2). Because all the 5 races were virulent to Koshihikari, which had been widely cultivated in Niigata area during the years, there were no drastic race changes. In addition, genetic variations in blast resistance indicated that Koshihikari also harbored the Pish gene, and that the Pia, Pii, and Pik genes were also dominant in the Hokuriku region, including Niigata Prefecture 21 . Virulent blast races against the resistance genes Pish, Pia, Pii, Pi3, Pi5(t), Pik, Pik-s, and Pi19(t) were dominantly distributed in Niigata Prefecture 22 . These reports confirmed that Koshihikari had been susceptible to dominant blast races before KO-BL introduction.
In the 2005 release year of KO-BL, the predominant blast races, 001.0 (virulent to Pik-s) and 003.0 (virulent to Pik-s and Pia), drastically decreased in distribution from 41.8% to 22.3% and 27.6% to 17.3%, respectively (Fig. 2a, Supplementary Table S2). Interestingly, races 001.0 and 003.0 rapidly decreased by 5.4% and 1.3% in 2006, respectively, even though especially Pia, which can be infected by the race 003.0, was used in the KO-BL composition. Because all ILs in the composition of KO-BL were resistant to race 001.0, and race 003.0 was only virulent to Pia, which made up 10% of the annual KO-BL composition (Table 1). In contrast, races 007.0 (virulent to Pik-s, Pia, and Pii) and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) dominated from 2005 to 2019. The higher rate of race 007.0 detection was affected by 30% of the ILs composing the annual KO-BL were susceptible. The second highest rate of race 037.1 detection was affected by a number of factors: the high susceptibility of a minor cultivar that had Pii and Pik, the mosaic configuration of fields typical in Niigata, and the air-borne spread of race 037.1. To maintain consensus on KO-BL cultivation based on total blast suppression in Niigata, rarely detected races virulent to resistant ILs in commercial fields are strictly supervised by the prefectural government to avoid unnecessary confusion in Niigata residents.
In 2008, to mathematically support KO-BL composition changes, we developed a simulation software to estimate long-term blast race dynamics in multilines using a plant-pathogen coevolution system 23 Table S4). The average occurrence of leaf and panicle blast was 46.1% and 52.9% during the 1995-2004 period and 9.5% and 9.6% during the 2005-2019 period, respectively. This resulted in a blast suppression effect by 70% of the resistant composition in KO-BL. Current seed production fields are rarely contaminated with virulent races against resistant KO-BLs. This suggests that seed sanitation contributes to the suppression of virulent pathogen epidemics in multilines. In addition, induced resistance 24,25 may have no effect on the practical use of multilines. Rice plants were found to induce a resistance response when inoculated with avirulent races of blast (those that stimulate protective responses to virulent race attacks). As the detection of several races in one area is rare and blast occurrence tends to be low, conditions that induce resistance in field situations do not occur. Fungicide applications to control blast in KO-BL and other minor cultivars decreased by approximately one-third during the 2005-2019 period compared with 2004 (Fig. 3b, Supplementary Table S5). Thus, the commercial scale use of crop diversity is clearly effective for the environmentally friendly control of airborne diseases.
The optimum long-term solution for pathogen population control using genetic diversity includes multilines. Blast occurrence in KO-BL introduced in Niigata, and the theoretical value of blast suppression in KO-BL tested at small scales, were reduced by approximately 10% compared to that of monoculture plots [26][27][28] . Thirty percent of susceptible ILs in KO-BL have the potential to improve compatible races with susceptible ILs and become predominant in large areas. This would contribute to the suppression of rapid increases in new virulent races emerging in the blast population. To maintain consensus on KO-BL cultivation based on total blast suppression in Niigata, rarely detected races virulent to resistant KO-BLs in commercial fields are strictly monitored by the prefectural government. Educating Niigata farmers ensures the long-term use of KO-BL. In fact, lower blast occurrence has been attributed to careful KO-BL cultivation and seed management.
The implementation of genetically diversified homogeneous seed mixtures, rotations with resistant KO-BL, restricted KO-BL cultivation, and pathogen monitoring allowed rice quality to be maintained, diseases to be suppressed, and environmentally sound agriculture to be economically viable in Niigata. Collaboration among   www.nature.com/scientificreports/ Our model also calculated a greater than 50-year persistence in terms of the small area effect in both prefectural cases. This result depends on an insufficient pathogen population increase in virulent mutations against resistant ILs (data not shown). In this way, the practical use of a multiline provides control without the need for as much fungicide with or without a periodic change in IL composition. Our results demonstrate that the management of crop and pathogen coevolution can control diseases at large scales and, thereby, contribute to global food security.

Methods
Rice plants. Rice  Blast-infected rice leaves and panicles were collected by Niigata prefectural officers under the Plant Protection Law from the Ministry of Agriculture, Forestry and Fisheries as part of the Japanese Government, and permissions were previously obtained from all farmers before sampling. The plant protection committees of all the Niigata municipalities were also authorized for these samplings.

Race investigation.
Our field tests were carried out in Niigata Prefecture (Japan), which has a wet season favorable for blast development during the rice growing season. To investigate blast race distributions, the Niigata area was divided into 5 km 2 grid squares. If leaf and panicle blast lesions were found in each grid square, infected leaf and panicle samples were collected by Niigata prefectural officers. Officers from the plant protection committees of the various Niigata municipalities also collaborated in the blast lesion sampling. Plant pathological research was performed using the single spore isolation method. The pathogenicity of isolated P. oryzae was determined by rice seedling inoculation using the race differentiation method 29,30 . Briefly, seedlings of the 12 Japanese differential varieties (Pik-s, Pia, Pii, Pik, Pik-m, Piz, Pita, Pita-2, Piz-t, Pik-p, Pib and Pit) at the 4-5 leaf age were sprayed with 1 × 10 4 spores/ml of the isolate and then moved to a humidic chamber (100% relative humidity) at 25 °C for 20 h. Inoculated plants were moved to a greenhouse at 25 °C for 1 week. Susceptible and resistant reactions were judged based on leaf blast lesion formation to determine the races of the isolates.
Disease assessments. Before each cropping season started, a representative field from each 15 km 2 grid square was selected to monitor disease and predict pest occurrence. After rice seedlings were planted in paddy fields, general epidemics, which initially started as a small number of leaf blast lesions, were observed in June. The subsequent development of leaf blasts (in July) and panicle blasts (in August and September) was evaluated as leaf blast lesion area per hill and diseased grains, respectively.

KO-BL composition determination.
Once a year, the Niigata prefectural committee assessed proposals for altering the resistant IL composition in KO-BL for the following two years using data on monitored race frequency and blast severity.
Simulation model. The gene-for-gene system 23 (Fig. 2b), mutation rate (10 -5 ) 31 , overwintering probability (0.01), favorable weather condition (10), maximum lesion number (10,000,000), number of simulated years (15), number of simulation trials (1000), and variables (lesion number of each race). Races on each IL increased exponentially until reaching the maximum lesion number, i.e., 10,000,000 × 10 (100,000,000) under favorable weather conditions every year. Mutated races emerged at a 10 -5 rate, and randomly selected races were overwintered, including mutated race from 10,000,000 × 10 × 0.01 (1,000,000) lesions. The 1000 trial results of the lesion number increase for each race were averaged in each year and transformed to the rates to show race dynamics.

Ethics declarations.
This study was performed in accordance with relevant rules, guidelines and regulations. There were no human subjects in this study.

Data availability
All the data used in the present study are included in this manuscript and supplementary information files; however, we will provide data from the simulation results for combinations of races and rice resistance genes, and all the raw data used in this study from the corresponding author upon reasonable request to all interested scientists. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.